JPS60180596A - Production of l-alpha-amino acid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention Field of Industrial Application The present invention relates to the general formula fI) C-OH HN-'O-H (wherein R is an aromatic group, a substituted aromatic group, an aliphatic group, or a substituted The present invention relates to a method for producing L-α-amino acids represented by (which is an aliphatic group).

In particular, the present invention relates to a method for enzymatically producing L-α-amino acids, wherein R in the general formula (i) is selected from 5C.

General formula CI) 'k! The compounds are useful as intermediates for the synthesis of compounds used in the pharmaceutical and chemical industries.

L-α-phenylalanine is an expensive product and is used in the synthesis of aspartame, which is useful as a sweetener.

PRIOR ART AND PROBLEMS Compounds of the general formula (I) are prepared by known methods, such as hydrolysis of proteins and isolation of amino acids from the reaction mixture.

However, the 7D1 method is uneconomical due to the use of expensive raw materials, the large number of processing steps required, and the overall low yield.

According to the invention described in Japanese Patent Publication No. 42-13850,
L-α-amino acids are hydantoins (5
-substituted hydantoin as a raw material, it is produced by the rIl method in the presence of an enzyme, and then separated and recovered from the reaction mixture.

Means to Solve the Problems The inventors discovered a simple and inexpensive method for producing L-α-amino acids, leading to the present invention.

According to the invention, the general formula fI) 1 C-OH HN-C-H, in which R is an aromatic group, a substituted aromatic group, an aliphatic group or a substituted aliphatic group, has the general formula laJ (Ill (wherein, R is the same meaning as above)
-Carbamyl-α-amino acid and L-N-carbamyl-
The α-amino acid form of N-carbamyl-α-amino acid was added in a liquid reaction mixture at a pH of 6.0 to 7.0 and a temperature of 20°C.
C to 40°C with an enzyme to form the general formula (
@ (wherein, D represented by R (ri has the same meaning as above)
- hydantoin compound and L-N-carbamyl-α-amino acid represented by general formula 11V) (wherein R has the same meaning as above); It decomposes and racemizes to produce D-N-carbamyl-α-amino acid and L-
N-carbamyl-α-amino acid 5, (c) On the other hand,
The L-N-carbamyl α-amino acid corresponds to
and (a) separating and recovering the L-α-amino acid from the reaction mixture.

Effect According to the method of the present invention, first, in step (a), D-N-carbamyl-α- represented by the general formula f) (wherein R is a group selected from the following) Amino acids and L-N-carbamyl-α-
The amino acid form of the compound was introduced into the liquid reaction mixture at pT(6,
0 to 7.0 and an enzymatic reaction at a temperature of 20°C to 40°C to produce a D-hydantoin compound represented by the general formula fl[) (wherein R has the same meaning as above) and the general formula 1y). (wherein, R has the same meaning as above)
-carbamyl-α-amino acid.

The enzyme used for this purpose is dihydropyrimidine hydrolase E, which is known in the art as having the ability to selectively hydrolyze hydantoins with asymmetric carbon atoms to produce D-N-carbamyl-α-amino acids. ,C03
, 5, 2, 2.

This enzyme can be obtained from extracts of animal organs or from Pseudomonas, Acromono (futa)
(7tchrOIIIO, baCter), Corynebacterium (COryneoaCterlum), Brevibacterium (Brevibacterium)
, Microbacterium
), Artrobacter
), Agrobacterium
+), Acrobacter,
Klebsiella, Sarcina, Protaminobacter
alflinODaoter), Streptomyces (
Streptomyces), Actinomyces (A
ctinomyces), Cjandi
da).

Rhodotorula, Pichia or Paecil
It is obtained from microorganisms belonging to the genus Omyces).

In a preferred embodiment of the invention, "Journal Off" by D. P. Wallah and S. Grisolia.
ノ (Iological Chemistry (J, Biol, Oh
Enzymes extracted from calf liver and enzymes extracted from the following microorganisms are used according to the method described in J. Em., J226.277 (1957).

AgeyZtrium Love Ionophther (A, rad
iobacter) NRRL B 11291/qy
Res vis (B, brevis) NRRL B
NRRL
B 11079 Pseudomonas (P, sp) CBS 1457
5 Pseudomonas (p, sp) CjBS
14675 Pseudomonas (P, sp) AT
CC11299εa-t'w;ζΦfunoset 4 cases (P
, fluoresaens) ATac 11250 system
a) ATCO12633 sheet U view sp'! Lytica (P, dea+IIolytica) N0
IB 8859 On the taxonomic characteristics of the above-mentioned microorganisms, M.

J, Pe1czav K YoP) rManual φ off, Microbiological φ methods (Manual
of Microbiology Method
Bergey's Manual of Determinative Bacteriology.
Determinative Bacteriolo
gy) 8th edition, 1974.

The microorganism is cultured under aerobic conditions in a liquid culture medium containing a nitrogen source, a carbon source, a phosphorus source, and a salt-free source.

Suitable carbon sources for this purpose are gelcols, charred hydrates such as sucrose, organic acids, alcohols and hydrocarbons.

The nitrogen sources used in the culture medium are organic and inorganic ammonia salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, urea and ammonia.

The inorganic salts used in the invention are potassium phosphate, sodium phosphate, magnesium sulfate, calcium carbonate, ferrous sulfate, magnesium sulfate and zinc sulfate.

A culture medium having the above composition was prepared using pT(,5,5 to 9.
Maintain between 0 and 20°C to 40°C.

Typically, cultures are grown at neutral pT (range, temperature 30°C and 2
It will be held in 4 hours.

Under suitable growth conditions, the microorganism produces within its cells an enzyme that converts said compound (ff) into the compounds (sou and i11').

The microbial boxes are generally separated from the culture medium according to known methods (e.g., centrifugation), followed by i'[I
i is contacted with compound (II) in the reaction mixture.

Alternatively, after separation from the culture medium, a rotary disintegrator, a French-pressure
, the bacterial cells are treated with ultrasonic waves, etc., and the protein fraction separated according to a conventional method is used for the enzymatic reaction of compound +II).

Furthermore, the technical and economic improvements of the present invention are realized by the Ita! J
This is accomplished by immobilizing the enzyme or enzyme-containing microorganism within the fibrous structure, as described in IFm% Patent No. 836,462. .

The amount of bacterial cells used in the enzymatic reaction of compound fII) is generally at a weight ratio of bacterial cells/compound fIIl of Aθ to 1/1.
00. This enzyme reaction is preferably carried out at a Fi temperature of 30°C, a pH of 5.5, and a reaction time of 24 hours.

By operating under such conditions, the compound (T[0 predominates as a solid, compound flit) is dissolved as a solution.

The temperature of such a dispersion is adjusted to a range of 0 to 10°C, and the pH of the dispersion is adjusted to a range of 24.0 to 5.5.
By adjusting within the range of , the compound (@) is completely or substantially completely precipitated.

In step (0) of the present invention, known techniques can be used for the separation and hydrolysis of the compound (IIO).

Generally, a compound is separated by filtration, centrifugation or other suitable means, while hydrolysis of this compound
In the liquid phase, in the presence of IN sodium hydroxide, at a temperature of 90 °C
In the reaction time of about 10 hours, in the case of engineering S (C), L-
The conversion of N-carbamyl-α-amino acid +11′) into L-α-amino acid II) is carried out according to known methods.

For the compound (ry) present in the solution, after removing the D-hydantoin compound, the compound flV) /
+ Molar ratio of nitric acid to 4, temperature 30°C 1 reaction time 4
Contact with nitric acid for 5 to 5 hours.

Alternatively, the conversion at step ic) involves the subsequent precipitation of compound (IV) from the solution obtained at step fb),
This will take place after 4 minutes. After removing the bacterial cells, compound (IV) is precipitated by adjusting the temperature to 10''C and pH to 3.1, and is isolated by methods known in the art.

In either case, after the transformation reaction, the produced L-α-
Amino acids are separated and recovered.

In a preferred embodiment of the invention, L-N-carbamyl-α-
The conversion reaction of the amino acids to the corresponding L-α-amino acids and the separation of the L-α-amino acids iI) are carried out in a single step.

That is, L-N-carbamyl-α-amino acid (IV)
and sodium nitrite is fed to a chromatographic column packed with acidic ion exchange tree 11, and the solution is allowed to ring for 4 to 5 hours. L- produced during the reaction
α-amino molecules) are fixed to acidic groups on the tree.

The L-α-amino acid is then recovered by dipping into a 2N* aqueous ammonium oxide solution. By shrinking the eluted melt, drying it, and neutralizing it, an optically subframe L is formed.
-α-amino acids are obtained.

The following examples are intended to illustrate the invention:
This is not intended to limit the invention.

Example 1 A culture broth with the following composition was prepared.

Composition Meat Begton 5z Kyo Extract 3? Glucose 5z Water 1 stone p) + 7.2 100yxJ of the above culture broth was dispensed into a circular Erlenmeyer flask (volume 0.5n) and sterilized at 110°C for 30 minutes.

Pseudomonas ass 1457 in a vertebral flask with
A culture of 5 stock bacteria was inoculated in an amount corresponding to that collected by Pt@Hawk. This culture has the above composition of 1 to 20% and agar (orFco) of 20%. //? The cells were pre-cultured for 24 hours at 30°C on a slant medium solidified with.

The thus inoculated flask was maintained at 30° C. for 24 hours under shaking (220 rpm+).

At the end of this time, the culture broth 1d was harvested under sterile conditions and transferred to a conical Erlenmeyer flask (g volume 0.5-6) containing 100 m/ml of culture medium of the above composition.

The round flask was maintained at a temperature of 30°C for 24 hours.

After this time, the solids were separated from the reaction mixture by centrifugation, and an equal volume of saline (NaCf f3y-1 water 1
! ).

After centrifugation, the compound DL-N-carbamylphenylalanine (R=g)-CH2 was dissolved in 50WL of water.
In a solution of pH 6.5 containing J 38t, cap body 20
0'# was suspended.

This suspension was stirred for 24 hours at a temperature of 30''C.

After this time had elapsed, a dispersion was obtained. The dispersion was adjusted to a temperature of 10'C and a pH of 5.5 to completely precipitate the product.

Then, the precipitated compound was removed from P and washed with water. In this way, a certain amount of 3,6-D-benzylhydantoin was obtained. In addition, the identification was performed by IR%NMR and elemental analysis.

This D-benzylhydantoin f was hydrolyzed and racemized by treatment with 40 yd of IN NaOH aqueous solution at a temperature of 90''C for 10 hours, resulting in D-N
-carbamylphenylalanine and [L-N-carbamylphenylalanine producing saceta.

The racemic mixture thus obtained was recycled.

On the other hand, L-N-carbamylphenylalanine present in the solution was removed by removing D-benzylhydantoin and by centrifuging the bacterial cells at a temperature of 10'C and a pH of 3.1, followed by precipitation. The precipitate thus obtained was collected by F and dried to reduce the king temperature.

3.8 fP of L-N-carbamylphenylalanine was obtained. In addition, for its identification, IR, NMR, and elemental analysis were used, and optical rotation power [α] 25°' = 38.5
(Confirmed by C=1, 1lJlIJ [(,]).

Then, 3.8 ? of L'-N-carbamylphenylalanine in 100 mJ of water? -< 18.3 mol)? Contain'f
In the Kf suspension, Na, N021.6'g-(23,
8 mmol) was added. Subsequent addition of IN aqueous ammonia solubilized the suspension.

Ion exchange resin (Amberlite IR120)
Chromatographic column packed with 30 g ('ui diameter 2
0 mm, length 30 rran), supplying the @ solution,
It was circulated for 4 hours. During the reaction between L-N-carbamylphenylalanine and nitrous acid, L-α-phenylalanine is produced, and this product is adsorbed onto the resin.

After the reaction was completed, the column was washed with water, and L-α-phenylalanine was eluted with a 2N aqueous ammonium hydroxide solution.

Then, the M eluate was concentrated and dried.

was gotten.

Example 2 DL-N-carbamylvaline as raw material Example 1
It was operated as follows.

The enzyme reaction was carried out at a temperature of 30°C for 124 hours, and the dispersion was cooled to 10°C and the pH was adjusted to 5.5.
Inglovirhydantoin 2.3y- was isolated. Subsequently, L-N-carbamylvaline was converted and the corresponding amino acid was separated.

5%, L-α-valine with INF4(J) 1.42
1 was obtained.

Working Example 3 The procedure was as described in Working Example 1 with DL-N-carbamylleucine as the substrate.

After enzymatic reaction, o-2-methyl-propylhydantoin 4
．． 2z was H%. For the identification of this compound, IR, NM
R and elemental analysis were used.

L-N-carbamylleucine was then converted and the corresponding amino acid was separated.

Optical rotation power [α] 20°-+12.0 (c = 2.5.
L-α-leucine 2.75 y- with 1NHOJJ
was gotten.

Example 4 Enzyme catalysts include those described above and by P. Wallach and S.

The procedure was as in Actual Example 1 using enzyme acetone powder 21 obtained from calf liver according to the method of Grisolia.

Enzyme reactions were carried out at 30°C for 24 hours. The dispersion was cooled to a temperature of 10°C and the pH was adjusted to 5.5.
-Benzylhydantoin 3.2y- was separated.

L-N-carbamyl-phenylalanine was then converted to the corresponding amino acid, resulting in L-α-phenylalanine 2.68 with an optical rotation power of 20° [α)o 34-1 (c = 1-94, water). % get leta.

Continuation of page 1 ■Int, C1,' identification symbol 0 shots clearer Felice Cenci Internal office reference number

Claims (1)

[Claims] 1 General formula fl) L represented by O-OH HN-0-H (wherein R is an aromatic group, a substituted aromatic group, and an aliphatic group is a substituted aliphatic group) - In the method for producing an α-amino acid, ial is represented by the general formula (111 (wherein, l has the same meaning as above) and 6 D
-N-carbamyl-α-amino acid and -N-carbamyl-α-amino acid in the form of -N-carbamyl-α-amino acid in a liquid reaction mixture at pH 6.0 to 7.0.
, a D-hydantoin compound represented by the general formula (Ill) (wherein R has the same meaning as above) and the general formula 1 by contacting with an enzyme at a temperature of 20°C to 40°C.
1V) L-N-carbamyl-α-amino acid represented by (wherein R has the same meaning as above), (b) separating the hydantoin compound from the reaction mixture and hydrolyzing it, Racemized to [)-N-carbamyl-
α-amino acid and L-N-carbamyl-α-amino acid, +cl; Meanwhile, the L-N-carbamyl-α-amino acid is converted into the corresponding L-α-amino acid, and (a) from the reaction mixture, L- A method for producing L-α-amino acids, which involves separating and recovering α-amino acids. 2. In the μs method described in claim 1, R in the general formulas fI), +II), (tU) and flIQ is selected from the following groups, L-α
-Production method of amino acids. 3% allowance The method for producing L-α-amino acids according to claim 1, wherein the enzyme used in step fa) is dihydropyrimidine hydrolase. 4. The method for producing L-α-amino acids according to claim 3, wherein the enzyme is obtained from calf liver. 5. The method for producing L-α-amino acids according to claim 3, wherein the enzyme is produced by a microorganism. 6% allowance In the manufacturing method according to claim 5, the microorganism is Pseudomonas (Pseudomonas a.
s) A method for producing L-α-amino acids belonging to the genus. 7 @ In the manufacturing method according to claim 6, the microorganism is selected from the following preserved microorganisms,
-Production method of α-amino acid. Pseudo+nonas OBS
L4575 Pseudomonas
s) CBS 14675 Pseudmonas
o+1onas) ATOC11299 Pseudomonas NCIB
8859des river olytica)'
omonas ATOO11250fluoresce
ns) 8. The method for producing L-α-amino acids according to claim 5, wherein the microorganism belongs to the genus Bacillus. 91m In the manufacturing method described in claim 8, the copying organism is Bacillus brevis.
) NRRL B 1180 Notilus sp'Cff
- Namofinomin Lsu (shiacillus 5tear)
omohe+uophi 1m5) NRRL B 110
A method for producing an L-α-amino acid selected from 79. 10 In the production method according to claim 5, the microorganism is Agrobacterium.
A method for producing L-α-amino acid, which belongs to umJ Akebono. 11. In the manufacturing method according to claim 10, the microorganism is Agronothylium nigra.
cteriuII+ NRRL B 1. ], 29] r
adiobacter), a method for producing L-α-amino acids. 12. In the manufacturing method according to claim 1, the enzyme reaction in step (a) is carried out at a temperature of 30°C and a pH of 6.
, 5. A method for producing L-α-amino acids. 13'# The method according to claim 1, wherein the hydrolysis in the step [bl is carried out in the presence of 1N NaOH in a liquid phase at a temperature of 90°C and a reaction time of 10 hours, L- Method for producing α-amino acids. 14% In the method # described in claim 1, the reaction in step 8 (C) below is carried out at a temperature of [30°C] using nitric acid.
A method for producing L-α-amino acids using C and a reaction time of 4 hours. 15. A method for producing L-α-amino acids, in which the separation in the step (dl) is performed by treatment with an ion exchange resin in the method for producing Tokuwakaori Seisui Range 1 Kamigai 1 Kaibi.